The invention relates to the use of acridine or acridine derivatives, preferably in combination with benzalkonium chloride, for the inactivation of enveloped or nonenveloped viruses. The process according to the invention is preferably carried out in the presence of proteins whose biological activity is largely retained.
It has been known for years that untreated human plasma or serum can contain human pathogenic viruses, such as HIV, HBV or HCV, which, if transmitted to sensitive recipients, can cause serious diseases, such as AIDS or hepatitis. In order to prevent this potential virus transmission, therapeutics which are obtained from human plasma or serum are prepared, on the one hand, only from preselected starting materials which, as far as anyone can judge, are virus-free and, on the other hand, are subjected to virus-inactivating/eliminating steps in the preparation process. The efficiency of the virus inactivation/elimination method used is established using strict measures and continuously checked.
Besides physical virus inactivation steps, chemical virus inactivation steps are also known in the preparation of said therapeutics. A particularly frequently discussed chemical process is the SD (solvent/detergent) method. It is suitable for inactivating enveloped viruses, i.e. viruses which are surrounded by a lipid-containing membrane, but has the crucial disadvantage of being completely ineffective against all known nonenveloped (uncoated) viruses. Moreover, also no other chemical process is known which would be suitable to inactivate nonenveloped viruses while simultaneously retaining the biological activity of the protein constituents of the therapeutic or of the human plasma or serum.
Although the chemical virus inactivation processes are only used in a supplementary capacity to the physical methods, and although most viruses potentially transmissible by blood and blood products carry a lipid coat, for reasons of safety there is an exceptional need to make available chemical inactivation processes which also reliably inactivate nonenveloped viruses. This is all the more desirable as recently also HAV and parvoviruses, for example parvovirus B 19, were discussed as viruses which are potentially transmissible by blood fluids or blood products. (Vox Sanguinis 67, Supplement 1, 1994: Proceedings of a Symposium held at the New York Blood Center).
The invention was also based on the object of developing an industrially utilizable process for chemical virus inactivation, in which enveloped and nonenveloped viruses, e.g. parvo viruses, are inactivated with retention of the biological activity of proteins present, e.g. therapeutically useful proteins.
This object is achieved according to the invention by adding acridine or an acridine derivatives to the protein-containing liquid to be treated. It was surprisingly found that acridine or acridine derivatives inactivate enveloped and nonenveloped viruses. Acridine derivatives are, for example, ethacridine, 9-aminoacridine (=amina-crine), 3,6-acridinediamine (proflavine), acrisorcin, Acrizane chloride (=phenacridane chloride), Acridine Orange, quinacrine, acricide, acridone, acridine-9-carboxylic acid, acranil (1-[(6-chloro-2-methoxy-9-acridinyl)amino]-3-(diethylamino)-2-propanol dihydro-chloride), 3,7-diamino-5-phenylphenazinium chloride (phenosafranin, Safranin B Extra), phenoxazine, phenothiazine and especially acriflavine (3,6-diamino-10-methylacridinium, chloride and 3,6-acridineidiamine) and their salts, e.g. chlorides, sulfates, bromides.
Surprisingly, it was additionally found that a combination of acridine or an acridine derivative and benzalkonium chloride displays a synergistic action during virus inactivation, i.e. the magnitude of the virus inactivation of the combination is higher than that of each individual substance.
The virus inactivation process according to the invention can be carried out with protein solutions such as blood, serum, plasma, blood products, allantoic fluid or milk. The virus inactivation is carried out at a pH of 3 to 10 or 5 to 9 (in, for example, serum or plasma solutions) and a temperature of 1xc2x0 C. to 80xc2x0 C., preferably of 20xc2x0 C. to 60xc2x0 C., very preferably from 20xc2x0 C. to 40xc2x0 C. or 25xc2x0 C. to 37xc2x0 C., and lasts 30 minutes to 10 hours, preferably 2 to 5 hours. For virus inactivation, a concentration of 1.0 g/l-0.00001 g/l or 1.0 g/l-0.004 g/l, preferably 0.1 g/l-0.001 g/l, is used for the acridines or acridine derivatives, and a concentration of 0.1 g/l-0.004 g/l, preferably 0.05 g/l-0.01 g/l, for benzalkonium chloride.
The removal of the acridines and of the benzalkonium chloride from the protein solutions, if this is necessary, is possible by means of simple, known methods, such as adsorption on active carbon or dialysis.
A further advantage of the virus inactivation process according to the invention is the very extensive protection of the protein constituents of the material to be treated: different biological activities, e.g. antibody activity and clotting-activity, are not reduced or only reduced to a tolerable extent.
The virus inactivation process according to the invention can therefore be employed, for example, for the decontamination of the following materials:
protein-containing solutions (dilute or concentrated)
blood or blood products; both the liquid and the cellular constituents
serum, plasma
allantoic fluid
organ extracts
milk
buffer solutions
antigens for diagnostics
vaccines, antigens for vaccines
The process according to the invention is furthermore suitable for disinfection in virus contamination of, for example, areas, equipment, effluents, wastes and surfaces of all types. The disinfection of virus-contaminated organ transplants, e.g. cornea, cerebral meninges, liver, heart, lungs or kidneys is also possible.